Most of the experiments during the first cycle of this MERIT Award were performed on isolated, perfused organs. The rationale for using perfused tissues initially was that each could be studied in isolation in a NMR tube so that if 13C signals appeared from unanticipated products of a HP-substrate, we could be confident that such metabolites were generated in that tissue and did not appear as a result of in vivo circulation between organs. Building on those experiments, we are now ready to translate our most interesting probes to image metabolism in real time in vivo as quickly as possible. We will focus on four aims: 1) measure glucose production by hyperpolarized 13C imaging in isolated perfused mouse livers and animals in vivo. This technology has important implications in better understanding the tissue sources of glucose production in diabetic animals and humans; 2) measure flux through the oxidative portion of the pentose phosphate pathways (PPPox) in vivo using HP-[1-13C]gluconolactone in injured tissues. Increased PPPox flux has been observed in times of cellular stress such as patients suffering from traumatic brain injury, ischemic heart disease, and cancer. Thus, given our discovery that HP-gluconolactone appears to be a selective probe of PPPox and there is currently no other simple way to measure PPPox in vivo, we believe this could lead to an important diagnostic tool for detecting damaged tissues by metabolic imaging; 3) We now know that kinetic conversion of HP-acetoacetate to HP-betaHB appears to be slow in healthy perfused tissues, an unexpected finding. Given that chemical exchange between these two species should be fast on the time scale of a hyperpolarization experiment like that seen for conversion of HP-pyruvate to HP-lactate, we feel it is important to fully understand the limitations of this reaction in healthy tissues versus tissues bearing damaged mitochondrial. If conversion occurs more rapidly in damaged mitochondria, this may provide a quick, useful, imaging assay for assessing tissue viability in vivo. 4) Finally, we will develop a polarization method to simultaneously measure extracellular pH and tissue necrosis using a mixture of HP-[1,4- 13C]maleic acid and HP-[1,4-13C]fumarate in cirrhotic livers in vivo. It has been shown that HP-fumarate is converted to HP-malate only in necrotic tissues because the dicarboxylate, fumarate, cannot be transported into most healthy cells in vivo. Given our discovery that HP-[1,4-13C]maleic acid should provide a direct readout of tissue pH from its chemical shift, we will explore the combined use of HP-[1,4-13C]maleic and fumaric acids as imaging probes of cell necrosis and extracellular pH in various models of damaged tissues